The goal of our studies is to characterize the function of a novel family of transmembrane ubiquitin ligases, particularly with respect to their role in regulating the immune response. Upon overexpression, members of the membrane-associated RING-CH (MARCH) protein family ubiquitinate the cytoplasmic tail of MHC I, CD4, B7.2 and other immunologically important cell surface molecules. As a result of ubiquitination, these target molecules are sorted to late endosomal multi-vesicular bodies and destroyed in lysosomes. Therefore, we hypothesize that ubiquitination plays an important role in regulating the constitutive or induced removal of these and other proteins from the surface of cells of the immune system. As an initial test of this hypothesis, we propose to study the role of ubiquitination during the constitutive endocytosis and degradation of MHC I molecules as well as during the lysosomal degradation of CD4 molecules upon T cell activation. We will further examine if MARCH proteins are involved in this ubiquitin-mediated sorting process by using gene knockdown technology. Moreover, we will use a proteomic approach to define the spectrum of membrane proteins regulated by this new family of ubiquitin ligases. During this exploratory period, we anticipate to gather sufficient evidence in support of a crucial function of MARCH proteins in cells of the immune system which will ultimately enable us to study MARCH protein function in vivo.